Shumborski Sarah J, Samuels A Lacey, Bird David A
Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
Department of Biology, Mount Royal University, Calgary, AB, T3E 6K6, Canada.
Planta. 2016 Oct;244(4):843-51. doi: 10.1007/s00425-016-2549-8. Epub 2016 May 28.
The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as 'amorphous,' lacking in ultrastructural features, and is often observed as a thin (~80-100 nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.
通过电子显微镜观察，拟南芥的角质层主要由角质/角质烷基质组成。角质层具有复杂的亚结构，这与角质层内蜡质的存在相关。植物角质层由不溶性聚酯角质和有机溶剂可溶性角质层蜡质组成，这些蜡质嵌入角质基质并覆盖其表面。这些成分在角质层中是如何排列的，目前还不太清楚。拟南芥的角质层通常被认为是“无定形的”，缺乏超微结构特征，并且经常被观察到是细胞壁表面上一层薄的（约80 - 100纳米）电子致密层。为了更详细地研究这种角质层，我们检查了茎快速伸长和成熟部分的角质层，并使用传统化学固定方法和高压冷冻/冷冻置换（HPF/FS）比较了角质层的保存情况。我们发现HPF/FS制备方法揭示了一种复杂的角质层亚结构，在较老的茎中更明显。我们还发现角质层厚度随着发育而增加，这表明聚合材料在增加，可能是以不可水解聚合物角质烷的形式。在样品制备之前通过氯仿浸泡提取蜡质时，揭示了蜡质对角质层形态的贡献。总体而言，电子致密的角质层仍然可见，但角质层亚结构消失了。此外，蜡质缺陷突变体cer6的角质层也缺乏这种亚结构，这表明这些细微条纹依赖于角质层蜡质的存在。我们的研究结果表明，HPF/FS制备方法可以更好地保存植物角质层，同时也为拟南芥角质层的精细结构提供了新的见解。
